The effects of the arbuscular mycorrhizal fungusGlomus deserticola on growth of tomato plants grown in the presence of olive mill residues modified by treatment with saprophytic fungi

Olive oil extraction generates large amounts of olive mill residues (DOR) which may be used as fertilizer. The influence of arbuscular mycorrhizal (AM) on the phytotoxicity of dry olive residue (DOR) transformed with saprophytic fungi was studied. Aqueous extraction of DOR gave an (ADOR) fraction and an exhausted (SDOR) fraction, both of which had less phytotoxicity for tomato than the original DOR. The saprophytic fungiTrametes versicolor andPycnoporus cinnabarinus further decreased the phytotoxicity of ADOR and SDOR on tomato. The decrease of phenols concentration and the differences in the level of laccase activity caused by these fungi suggest did not account fully for the reduced phytoxicity but the fact that the higher hydrolytic enzyme activity ofP. cinnabarinus, paralleled the decrease of phytotoxicity, indicates that these enzymes seem to be involved. The AM fungusGlomus deserticola increased or exacerbated the beneficial effect of SDOR incubated with saprophytic fungi, in terms of dry weight of tomato plants. The percentage of root length colonized byG. deserticola strongly decreased in presence of DOR, but the level of mycorrhization was higher in presence of ADOR or SDOR. Our results suggest that the combination of aqueous extraction and incubation with saprophytic fungi will open the way for the use of olive oil extraction residues as organic amendment in agricultural soils.     

Phenolic removal of olive-mill dry residues by laccase activity of white-rot fungi and its impact on tomato plant growth

We studied the influence of the laccase activity of two white-rot fungi on the toxic effect of water-soluble substances from dry residues of olives (ADOR) on tomato plants. Pycnoporus cinnabarinus and Coriolopsis rigida decreased the phenol content of ADOR to 73% after 15 days. P. cinnabarinus and C. rigida produced laccase activity after 5 and 15 days, respectively, and the highest activity in both fungi was detected at 20 days. The treatment of ADOR with these white-rot fungi decreased the phytotoxicity of this residue on tomato plants. A close relationship was found between the amount of laccase produced, the decrease in phenol content of ADOR by the saprobic fungi, decrease of phytotoxicity of ADOR, and the increase in dry weight of tomato plants. These results show that phenol removal by the laccase activity of white-rot fungi can be important in the elimination of phytotoxic substances present in olive-mill dry residues.     

Oxidative Stress Induced in Sunflower Seedling Roots by Aqueous Dry Olive-Mill Residues

The contamination of soils with dry olive-mill residue can represent a serious problem as being an environmental stressor in plants. It has been demonstrated that inoculation of aqueous extract of olive oil-mill residue (ADOR) with saprobe fungi removes some phenolic compounds. In this paper we studied the effect of ADOR uninoculated or inoculated with saprobe fungi in sunflower seedling roots. The germination and root growth, O₂·^- generation, superoxide dismutase (SOD) and extracellular peroxidases (EC-POXs) activities, and the content of some metabolites involved in the tolerance of stress were tested. The roots germinated in ADOR uninoculated show a decrease in meristem size, resulting in a reduction of the root length and fresh weight, and in the number of layers forming the cortex, but did not alter the dry weight, protein and soluble amino acid content. ADOR caused the decreases in O₂·^- generation and EC-POX′s activities and protein oxidation, but enhanced SOD activity, lipid peroxidation and proline content. Fluorescence imaging showed that ADOR induced O₂·^- and H₂O₂ accumulation in the roots. The increase in SOD and the decrease in EC-POX′s activities might be involved in the enhancement of H₂O₂ content and lipid peroxidation. Control roots treated with ADOR for 10 min show an oxidative burst. Roots germinated in ADOR inoculated with saprobe fungi partially recovered normal levels of ROS, morphological characteristics and antioxidant activities. These results suggested that treatment with ADOR caused a phytotoxic effect during germination inducing an oxidative stress. The inoculation of ADOR with saprobe fungi limited the stress.     

Interactions between phenolic compounds present in dry olive residues and the arbuscular mycorrhizal symbiosis

The use of “alpeorujo” (dry olive residue) has been proposed as an organic amendment in order to enhance soil structure and to increase C storage in soils. The aim of this work is to study how aqueous alpeorujo (ADOR) extracts bioremediated with white-rot fungi and three representative phenolic acids present in this extract (protocatechuic, vanillic and caffeic acid) affect the growth of the arbuscular mychorrhizal fungus Rhizophagus custos in monoxenic culture. Our results show that ADOR decreased mycorrhization parameters; however, this negative effect ceased after ADOR bioremediation. Although protocatechuic and vanillic acids have drastic negative effects at high concentrations, these phenols enhance mycorrhization processes at low concentrations and caffeic acid negatively affects symbiosis at low concentrations. Finally, the capacity of root biomass to dissipate individual phenols was also estimated, in which mycorrhized roots improve phenol dissipation in the growth medium in the presence of different phenols. This study highlights the important role played by arbuscular mycorrhiza in protecting plants from phytotoxicity.     

Hyphal growth and mycorrhiza formation by the arbuscular mycorrhizal fungus Glomus claroideum BEG 23 is stimulated by humic substances

Effects of humic substances (humic acid or fulvic soil extract) or saprophytic microorganisms (Paecilomyces lilacinus and an unidentified actinomycete) on growth of mycelium and mycorrhiza formation by Glomus claroideum BEG23 were studied in a hydroponic system. Humic substances stimulated root colonization and production of extraradical mycelium by the mycorrhizal fungus. Both humic and fulvic acids tended to decrease populations of culturable bacteria and fungi in the cultivation system, indicating a moderately antibiotic activity. The addition of saprophytic microorganisms able to use humic substances to the cultivation system further stimulated the development of the mycorrhizal fungus. However, stimulation of G. claroideum was also observed when the saprophytic microorganisms were heat-killed, suggesting that their effect was not linked to a specific action on humic substances. The results indicate that humic substances may represent a stimulatory component of the soil environment with respect to arbuscular mycorrhizal fungi.     

Production of plant protoplasts by enzymes from selected wood-destroying fungi

The goal of this work was to select higher fungi for the production of enzymes able to produce plant protoplasts. The hydrolytic capacity of various species of wood-detsroying fungi was examined to select those with the maximal hydrolytic capacity. The selection criteria included attack on cellulose, and oligo-and polysaccharides by each fungus species. Lenzites elegans, gonoderma applanatum and Pycnoporus sanguineus produced thr richest enzymatic mixtures, which were evaluated for their protoplasting ability. L. elegans has the best enzymatic mixture able to [roduced plant protoplasts with the shortest time for maceration of plant tissues, followed by G. applanatum and P. sanguineus. The potential of these fungi and the simplicity of enzyme proudtcion suggest the possibility of commercial applications.Correepondence to: A. R. Sampietro     

Synergism between fungal enzymes and bacterial antibiotics may enhance biocontrol

The interactions between biocontrol fungi and bacteria may play a key role in the natural process of biocontrol, although the molecular mechanisms involved are still largely unknown. Synergism can occur when different agents are applied together, and cell wall degrading enzymes (CWDEs) produced by fungi can increase the efficacy of bacteria. Pseudomonas spp. produce membrane-disrupting lipodepsipeptides (LDPs) syringotoxins (SP) and syringomycins (SR). SR are considered responsible for the antimicrobial activity, and SP for the phytotoxicity. CWDEs of Trichoderma spp. synergistically increased the toxicity of SP_25-A or SR_E purified from P. syringae against fungal pathogens. For instance, the fungal enzymes made Botrytis cinerea and other phytopathogenic fungi, normally resistant to SP_25-A alone, more susceptible to this antibiotic. Pseudomonas produced CWDEs in culture conditions that allow the synthesis of the LDPs. Purified bacterial enzymes and metabolites were also synergistic against fungal pathogens, although this mixture was less powerful than the combination with the Trichoderma CWDEs. The positive interaction between LDPs and CWDEs may be part of the biocontrol mechanism in some Pseudomonas strains, and co-induction of different antifungal compounds in both biocontrol bacteria and fungi may occur. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of the saprophytic fungus Fusarium oxysporum on arbuscular mycorrhizal colonization and growth of plants in greenhouse and field trials

Effects of the saprophytic fungus Fusarium oxysporum on arbuscular mycorrhizal (AM) colonization and plant dry matter were studied in greenhouse and field experiments. Host plants: maize (Zea mays L.), sorghum (Sorghum vulgare L.), lettuce (Lactuca sativa L.), tomato (Lycopersicum esculentum L.), wheat (Triticum vulgare L), lentil (Ervum lens L.) and pea (Pisum sativum L.), the AM fungi: Glomus mosseae, G. fasciculatum, G. intraradices, G. clarum, and G. deserticola and the carriers for F. oxysporum inoculum: aqueous solution, thin agar slices, and pellets of agar and alginate were tested under greenhouse conditions. Greatest plant growth and AM colonization responses in sterilized and unsterilized soils were observed with pea, Glomus deserticola and sodium alginate pellets as the carrier for F. oxysporum inoculum. Under field conditions, adding F. oxysporum increased the survival of transplanted pea, possibly through a beneficial effect on AM fungi. Application of F. oxysporum increased shoot dry matter, N and P concentrations of pea and sorghum plants, and the level of AM colonization attained by indigenous or introduced AM fungi. These parameters were similar in plants inoculated with either G. deserticola or with the indigenous AM fungi. Application of the saprophytic fungus increased the number of propagules of AM fungi in field plots in which pea was grown, but this increase was not sufficient to increase AM colonization of sorghum after the pea crop. This revised version was published online in June 2006 with corrections to the Cover Date.     

Conversion of crude chitosan to an anti-fungal protease by <Emphasis Type="Italic">Bacillus cereus</Emphasis>

Bacillus cereus AU004, isolated from soil samples, secreted a complex of hydrolytic enzymes into the culture broth when it was grown aerobically in a medium containing crude chitosan flakes. The presence of the AU004 culture supernatant substantially influenced the growths of the plant-pathogenic fungi Fusarium oxysporum, F. solani and Pythium ultimum in terms of dry weight. AU004 excreted a protease when cultivated in a medium that contained 4% (w/v) chitosan as the major nutritional source. The protease was purified by sequential chromatography and characterized as a novel extracellularly neutral protease. The protease had an Mr of 28.8 kDa. The optimal pH and temperature for protease activity were 7 and 50°C, respectively. Antifungal activity of the protease was observed using an assay based on the inhibition of spore germination and hyphal extension of the fungal Pythium ultimum. This investigation is the first report of the production of an anti-fungal protease from Bacillus spp.     

Decolourization of Municipal Effluent and Sludge by Pleurotus sajor-caju and Pleurotus ostreatus

Summary The Americana Municipal Treatment Station, S?o Paulo, Brazil, manages 400 l of effluent s⁻¹, from domestic and textile origin, which produces an average of 20 t of sludge per day. The decolourization of the effluent and sludge by three strains of Pleurotus (Pleurotus sajor-caju F2, F6 and Pleurotus ostreatus) was evaluated. The strains of P. sajor-caju F2 and F6 were able to decolourize the sludge, while P. ostreatus was less efficient. Detoxification was appraised with three bioassays comprising the cnidarian Hydra attenuata, the alga Selenastrum capricornutum and lettuce seeds. After exposure to fungi, effluent toxicity decreased but not that of its sludge. Strain P. sajor-caju F6 presented signs of toxicity shown by electron microscopy in the presence of the effluent. The three strains produced high amounts of manganese-peroxidase (Mn–P) and laccase in the presence of the sludge. Although P. ostreatus produced large amount of Mn–P and laccase enzymes, these enzymes did not result in decolourization of the sludge, suggesting that other factors are likely to be involved. Carbon content decreased only in the treatment with P. ostreatus.     

Effect of herbizid and touchdown herbicides on soil fungi and on production of some extracellular enzymes

Glucophilic and cellulose-decomposing fungi were significantly reduced in soil samples treated with 0.019-0.152 mg a.i./kg soil of the herbicides Herbizid and Touchdown. The decrease was regularly correlated with the doses of the two herbicides and persisted till the end of the experiment (12 weeks). The isolated fungi were found to be able to produce hydrolytic extracellular enzymes in solid media but with variable capabilities. The ability to produce enzymes was adversily affected by the incorporation of herbicides in culture media. Lower doses of herbicides were occasionally promotive to enzyme production and mycelial growth of some fungi. Incorporation of 50 ppm of Herbizid and Touchdown significantly activated amylase production and mycelial dry weight in cultures of Fusarium oxysporum, Mucor hiemalis and Penicillium chrysogenum. There was a significant increase in C1-cellulase produced by F. oxysporum and P. aurantiogriseum when cultures were treated with 50, 100 and 200 ppm of Herbizid which induced also more Cx-cellulase production by P. chrysogenum. Lipase and protease production was always lower in treated than in control fungal cultures.     

Colonization ofRetama raetam seeds by fungi and their significance in seed germination

Examination by scanning electron microscopy and incubation on potato-dextrose agar medium showed that dry seeds ofRetama raetam were externally free of fungi. When planted in sandy loam soil, the seeds become colonized with eleven soil-borne fungal species. The fungi were isolated on cellulose agar, pectin agar and lignin agar media.Aspergillus flavus, A. niger, A. fumigatus, Penicillium capsolatum andFusarium oxysporum had broad occurrence and were recovered on all the three media. The production of hydrolytic enzymes by the isolated fungi depends on the substrate and species.Penicillium capsolatum, P. spinulosum andA. niger had wide enzymatic amplitude and they were able to produce cellulolytic, pectolytic and lignolytic activities on corresponding substrates as well as on seed-coat-containing media. The lignolytic activities of the isolated species exceptChaetomium bostrychodes andTrichoderma viride were enhanced by applying the seed-coat materials as C- source rather than lignin. SoakingR. raetam seeds in culture filtrates of most of the fungi grown on seed-coat-supplemented media induced a pronounced and distinct stimulating effect on seed germination. The most effective filtrates were those ofP. capsolatum, P. spinulosum andSporotrichum pulverulentum.     

Production of polysaccharide-degrading enzymes by saprophytic fungi from glyphosate-treated flax and their involvement in retting

The fungi present on glyphosate-treated flax plants were isolated. Cladosporium herbarum, Epicoccum nigrum, Botrytis cinerea and yeasts occurred most frequently immediately after glyphosate treatment but as retting progressed the frequency of occurrence of Fusarium culmorum, Alternaria alternata and a Phoma sp. increased. Many of the fungi isolated from retting flax were also present as epiphytes on healthy flax stems. Glyphosate was shown to be fungitoxic in vitro but it had only a very slight effect on fungi colonising the flax. The application of sucrose and urea to flax 1 wk after glyphosate treatment resulted in more rapid fungal colonisation of the stems, but did not significantly enhance retting. When grown on sterilised flax stem sections, fungi known to be saprophytic on flax produced polysaccharide-degrading enzymes. All seven fungi tested produced polygalacturonase, pectin-lyase and xylanase. The greatest cellulase activity was present in stem tissues inoculated with F. culmorum and the Phoma sp. while no cellulase was detected in tissue inoculated with B. cinerea, a Mucor sp. or a Penicillium sp. Extracts from flax inoculated with the cellulolytic fungi caused the solubilisation of native cellulose. Pectinases, xylanase and cellulase were also detected in naturally-colonised senescing and dead flax stems. Stems which had been treated with a sucrose solution tended to contain the greatest enzyme activity.     

Mechanisms of coal solubilization by the deuteromycetes Trichoderma atroviride and Fusarium oxysporum

Three different mechanisms can be envisaged that are used by fungi to solubilize coal: the production of alkaline substances, the extrusion of chelators and, of special interest in the scope of biotechnology, the action of enzymes. Whether these mechanisms are operating separately or in various combinations has not yet been finally assessed. The two deuteromycetes Fusarium oxysporum and Trichoderma atroviride solubilize coal by synergistic effects of various different mechanisms depending on the cell metabolism. F. oxysporum seems to solubilize coal by increasing the pH of the mycelial surroundings and by the action of chelators induced during growth in glutamate-containing media (without involvement of enzymes). T. atroviride, on the other hand, appears to use, in addition to an alkaline pH and a high chelator activity, at least two classes of enzyme activity to attack coal: hydrolytic activity for coal solubilization and ligninolytic activity for degradation of humic acids. Received: 3 February 1998 / Received revision: 31 August 1998 / Accepted: 3 September 1998     

Hydrolytic enzyme activities of extracted humic substances during the vermicomposting of a lignocellulosic olive waste

Humic substances and three hydrolytic enzymes (beta-glucosidase, phosphatase and urease) were extracted by neutral sodium pyrophosphate from an olive waste (dry olive cake), alone or mixed with municipal biosolids, during a nine month vermicomposting process. Easily degradable compounds decreased during the vermicomposting process because of microbial consumption. When municipal biosolids were added to dry olive cake, microbial activity increased and the amounts of compounds extracted by pyrophosphate were three times lower than olive cake alone. In both instances, beta-glucosidase, phosphatase and urease activities of the organic extracts either increased or remained the same after a nine month period of vermicomposting, thus suggesting that the humus enzyme complexes resisted microbial and earthworm attack. It is known that humus immobilised enzymes also remain active in soil environments, reactivating the nutrient cycles in soil. The use as amendments of vermicomposted olive cake, alone or when mixed with biosolids, could be a good alternative to reactivate the C, P and N-cycles in degraded soils for regeneration purposes.     

Degradation of Raspberry Suberin by Fusarium solani f. sp. Pisi and Armillaria mellea

When submers cultures of Fusarium solani f. sp. pisi and Armillaria mellea were grown in a medium supplemented with 0.5 % suberin isolated from raspberry periderm, hydrolytic enzymes were produced and measured by a spectrophotometric assay using p-nitrophenyl butyrate as substrate. The enzymatic activity in the culture fluids reached its peak after 32 to 44 days of incubation. In a gas-chromatographic assay of the enzymatic degradation of suberin, concentrated culture fluids of suberin-grown fungi were incubated with raspberry suberin. The culture fluids of F. solani and A. mellea catalyzed the release of chloroform-soluble products, which were analyzed by gas-liquid chromatography. Suberin monomers like fatty alcohols and acids with chain-lengths from C₁₆ to C₂₆ as well as C₁₆ and C₁₈ω-hy-droxyacids could be identified as products. The suberin-induced enzymes showed catalytic properties similar to cutin-hydrolyzing enzymes previously isolated from different fungi.     

Effect of kind and method of fungicidal treatment of bean seed on infections by the VA-mycorrhizal fungus Glomus macrocarpum and by the pathogenic fungus Fusarium solani

To test the effect of seed treatment with fungicides on the development of mycorrhizal fungi, bean seeds were treated with fungicide dry or vehicled in the organic solvents, ethanol or dichloromethane and then planted in soil inoculated with the mycorrhizal fungus Glomus macrocarpum and/or the plant pathogenic fungus Fusarium solani. Measurements were made at 4 day intervals, to evaluate the location and extent of colonization of either Glomus macrocarpum or Fusarium solani in the root system. Most combinations of fungicide-solvent had little effect on the extent of colonization by each fungus individually. However, when both fungi were inoculated together, symptoms of F. solani were seen only in the tips of roots which indicate that the mycorrhizal fungus was able to limit the occurrence of the pathogenic fungus.     

Genetic and physiological response to fumonisin and AAL-toxin by intact tissue of a higher plant

The differential phytotoxicity of purified AAL-toxin to lines of tomato isogenic for the Asc gene parallels resistance to Alternaria alternata f.sp. lycopersici. This relationship, as reported earlier, is consistent with the role of AAL-toxin as a host-specific toxin with the role of a primary chemical determinant of Alternaria stem canker. Current results indicate the pathogen and the AAL-toxin also can be recovered from ripe fruit with symptoms of the disease known as black mold. Fumonisins are structurally similar to the AAL-toxins but are secreted by Fusarium moniliforme which is taxonomically distinct from A. alternata. F. moniliforme, is not pathogenic on living tomato tissues but was recovered from ripe tomato fruit with symptoms of black mold. The penetration of ripe fruit and subsequent colonization by both fungi appears to be saprophytic. Fumonisins and AAL-toxins express equivalent genotype-specific activity against the isogenic Asc lines of tomato and produce equivalent necrotic symptoms in tomato leaflet bioassays. Evidence was obtained that the biosynthetic pathway for production of these toxins is present in several species of both Alternaria and Fusarium. Toxin biosynthesis was sensitive to nutritional regulation in both genera. However, pathogenicity on tomato was not altered by the medium used for inoculum production in either genera and remained restricted to A. alternata f.sp. lycopersici in the studies reported here. Differences in the amount of toxin produced were found among isolates of both genera while the magnitude of the differences was defined by the substrate on which the fungi were grown.     

Antifungal activity of extracellular hydrolases produced by autolysing Aspergillus nidulans cultures

Carbon-starving Aspergillus nidulans cultures produce high activities of versatile hydrolytic enzymes and, among these, ChiB endochitinase and EngA ??-1,3-endoglucanase showed significant antifungal activity against various fungal species. Double deletion of engA and chiB diminished the antifungal activity of the fermentation broths and increased conidiogenesis and long-term viability of A. nidulans, but decreased the growth rate on culture media containing weak carbon sources. Production of ChiB and EngA can influence fungal communities either directly due to their antifungal properties or indirectly through their effects on vegetative growth. Our data suggest saprophytic fungi as promising future candidates to develop novel biocontrol technologies.     

Interrelationship betweenCoprinus lagopus andFusarium udum in soil in relation to competitive saprophytic ability and microbial antagonism

Summary The interrelationship between the growth ofCoprinus lagopus andFusarium udum on pigeon pea substrates in soil was studied at 22-2°C and 30±2°C in relation to their competitive saprophytic ability and antagonism.C. lagopus was observed to be a potent and frequent colonizer of pigeon pea substrates in soil along withF. udum. Saprophytic colonization of pigeon pea substrate byF. udum precolonized byC. lagopus was inhibited in all inoculum soil mixtures. Saprophytic colonization of the substrate byF. udum was better at 22°C than at 30°C and that byC. lagopus better at 30°C than at 22°C. The colonization of substrate by each fungus was suppressed by the other fungus depending upon the temperature used. Hyphal parasitism and colony interactions between these fungi and also with a number of other saprophytic fungi were investigated. No antagonistic reaction was observed betweenF. udum andC. lagopus. However, these fungi were observed to be potent antagonists against other microfungi tested. The formation of fruiting bodies ofC. lagopus was also observed in the laboratory on nutrient media but more frequently on pigeon pea substrates.